The long-term goal of this program is to understand how the quinolones kill Mycobacterium tuberculosis. The present proposal focuses on how these compounds behave in quinolone-gyrase-DNA complexes to cause chromosome fragmentation and rapid cell death. A goal is to identify structural features of the quinolones and gyrase that enhance lethal action, particularly with non-growing bacteria. Preliminary studies have identified structural moieties that make some quinolone derivatives exceptionally active at killing mycobacteria when protein synthesis is blocked, and work with other bacteria indicates that this lethality may arise from quinolone-induced, gyrase-mediated chromosome fragmentation. With new C-8-methoxy fluoroquinolones, neither chromosome fragmentation nor cell death requires ongoing protein synthesis, DNA replication, or aerobic growth, suggesting that these compounds may be able to kill non-growing M. tuberculosis. M. tuberculosis DNA gyrase, the molecular target of quinolones, will be purified and used to study biochemical interactions of quinolones with gyrase-DNA complexes formed with isolated chromosomes (nucleoids) and plasmids. Alteration of gyrase and quinolone structure will be used to probe the mechanism of chromosome fragmentation. Gyrase changes will focus on mutations expected to affect GyrA dimer interactions;quinolone variation will involve the quinolone core ring structure and substituents attached at the N-1, C-6, C-7, and C-8 positions. Chemical cross-linking will be used with ternary drug-enzyme-DNA complexes to characterize aspects of drug binding such as drug-gyrase orientation. Knowledge of how particular quinolone substituents destabilize drug-enzyme-DNA complexes and release lethal DNA breaks will be used to design new quinolones. The most active will be tested for the ability to kill cultured cells after growth has been halted by blocking protein synthesis, by gradual removal of oxygen, and by treatment with nitric oxide. Quinolones that are exceptionally active at fragmenting chromosomes in vitro and killing cultured cells will be examined for lethality with M. tuberculosis in a murine model of infection in which M. tuberculosis growth and growth arrest can be observed. This work is expected to provide information for the design of a new generation of quinolone characterized by rapid killing of non-growing bacterial cells, a property that may shorten treatment of tuberculosis and help limit multidrug-resistant tuberculosis.
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